Project Abstract Head and neck cancer represents the sixth most common malignancy worldwide, and oral squamous cell carcinomas (OSCC) are the most frequent malignancies in the oral cavity. Despite improvements in prognosis for many other human cancers, survival rates for OSCC have remained nearly unchanged for decades, emphasizing the urgent need for new treatment modalities. Mounting evidence indicates that the human microbiome plays a key role in promoting a variety of cancers, including stomach, colon and gall bladder, via various mechanisms. In contrast to the lower GI tract, the role of, and mechanisms by which, the oral microbiome may contribute to OSCC remain obscure. The impetus for this project is that gnotobiotic mouse models, coupled with functional transcriptomics, offer powerful new approaches to elucidate the mechanisms that underlie the tumorigenic effects of the oral microbiome in OSCC. In preliminary studies we found that germfree mice colonized with microbiomes, and challenged with the carcinogen 4-NQO, developed many more, and larger tumors than mice that remained germfree. Transcriptionally-hyperactive candidate pathogens were identified, which expressed numerous virulence signatures, paralleling previous findings in human OSCC. Tumor-infiltrating leukocytes (TIL) appeared to be modulated to an immunosuppressive vs protective phenotype in microbiome-colonized mice. These findings lead to our central hypothesis, that key pathogenic species in the oral microbiome promote OSCC progression, at least in part by inducing immunosuppression vs protective immunity. This will be tested by determining the transcriptionally-hyperactive species that most strongly promote the growth and metastasis of syngeneic mouse oral cancer (MOC) cell line orthografts in mouse tongues in vivo. Bacterial metabolic pathways will be characterized by metatranscriptomics, and the microbiome-modulated immune as well as tumor cell responses by RNASeq. Immune responses induced by tumorigenic pathogens will be characterized locally and in draining lymph nodes. The function of Treg and myeloid-derived suppressor cells will be evaluated by abrogation with immunotherapeutic antibodies in vivo. Direct effects of tumorigenic pathogens on tumor cell growth and invasion will be determined. The goal is to identify key oral tumorigenic bacteria and their molecular mechanisms that are responsible for promoting OSCC progression. Successful completion of this project will suggest new microbiome-focused interventions to reduce OSCC initiation and recurrence, and contribute to the development of next generation therapeutics for this recalcitrant and devastating disease.